I found that a longer mix kept carryover to a minimum on the BD instruments. I have also found that carryover still occurs at times. Sometimes from the mix or wash tubes themselves. We've also found a wash necessary before running any tube with rare cells, regardless of the mix time. In my experience at the clinical lab I used to work in, this extra time was a luxury we didn't have. I have also found that despite extensive attempts to educate investigators here in the research core lab, most seem to find it inconvenient, or unnecessary to properly flush the SIP when they run samples, let alone clean the instrument correctly. This phenomenon has proved to cause a larger problem with carryover. I guess my point is, I would do whatever it took to make any lab I were running the best it could be. Personally, I wouldn't be afraid of doing some comparison shopping to ensure that happened. I would hope that most of the flow people out there aren't so restricted to one side of the BD/Coulter fence that they'd be afraid to do that. I do understand how that happens, though. I've seen it first hand. Investigators afraid to use PC5 because they've read it's "messy", while using CyChrome or Tricolor in some of their assays. It's all the same thing. If a product is good, I say use it, no matter who makes it. Dax Arguello Huntsman Cancer Institute Flow Cytometry Core Facility Salt Lake City, UT dax.arguello@hci.utah.edu (801) 581-8641 -----Original Message----- From: Keith Bahjat [mailto:kbahjat@ufl.edu] Sent: Friday, September 24, 1999 3:05 PM To: cyto-inbox Subject: Re: Cell carryover on FacsCalibur with loader As I understand it, with no tube on the SIP, sheath fluid flow backwards out the SIP, where it would normally drip out, but is then caught by the DCM. Giving the instrument a couple of seconds with the DCM running should get enough sheath fluid backflushing to clear cells from the previous sample. Clinical labs utilizing cytometers are supposed to verify that insignificant carryover between samples occurs. To test your instrument, run a tube of PBS at various times after removing a tube contaiing stained cells. We found that the typical 3 to 5 second delay implemented by the loader was enough to flush sample from the SIP, but that sometimes users manually placing tubes on the SIP would not wait long enough for this backflush to complete. Additionally, when initially placing a sample tube on the SIP, a small amount of sheath fluid drips back into the tube. If you do not allow time for the SIP to flush fully, this drop will contain cells from the previous sample. I use a 10 second mix between tubes, and see no carryover. During intense investigation of this matter while running thiazole orange, I was unable to detect rbc in subsequent PBS samples. I had suspect RBC's would cling to the outer metal of the SIP, but they apparently did not. Keith Bahjat kbahjat@ufl.edu Keith Bahjat Graduate Assistant University of Florida College of Medicine Gainesville, Florida kbahjat@ufl.edu
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